C型肝炎病毒非結構性蛋白質NS4A抑制轉譯與 誘導病毒NS3蛋白質產生內部截切之作用機制

The Mechanisms of Hepatitis C Virus NS4A Protein on the Inhibition of Protein Synthesis and the Internal Cleavage of NS3 Protein

王怡敏(Yi-Ming Wang)

指導教授 ： 張鑫

國立臺灣大學/醫學院/微生物學研究所/碩士(2005年)

https://doi.org/10.6342/NTU.2005.01470

摘要

C型肝炎病毒 (Hepatitis C Virus，HCV) 為一單股正向RNA病毒。先前的研究已知病毒非結構性蛋白質NS3具有serine protease與helicase的生物活性，而其serine protease的活化需要非結構性蛋白質NS4A作為輔因子。NS4A會與NS3結合形成穩定的複合物，增進NS3 serine protease的切割作用，對於HCV非結構性蛋白質的成熟扮演重要的角色。近幾年的研究指出，NS4A會廣泛地抑制細胞與病毒的轉譯作用。本實驗室先前利用GST pull down的方法發現NS4A可藉中間區域與細胞內轉譯延伸因子eEF1A結合。本研究中，利用螢光酵素 (luciferase) 為報導質體，分別進行活體外轉錄轉譯反應及細胞轉染，證明NS4A抑制轉譯的作用是存在的，並更進一步定出eEF1A是利用羧基端與NS4A產生交互作用。另外，對NS4A進行胺基酸的點突變，以免疫沉澱法與西方點墨法分析，發現NS4A(V23A) 和NS4A(I25AV26A) 與eEF1A結合能力較NS4A野生株為弱，另外利用luciferase assay也觀察到NS4A(V23A) 與NS4A(I25AV26A) 對於抑制luciferase蛋白質的合成效果較野生株差；反之，NS4A(V24A) 與eEF1A的結合能力則與NS4A野生株相當，在抑制luciferase活性上的程度亦與NS4A野生株相當。eEF1A是轉譯進行的重要因子，依上述結果可推測，NS4A可能藉由與eEF1A的結合來影響轉譯的進行。
另一方面，先前Yang等人的研究顯示，在一些哺乳動物的細胞中 （HepG2、COS-7和NIH3T3），輔因子NS4A存在時會活化NS3 serine protease，繼而促使NS3被截切為更小的片段，稱之為NS3的內切片段。在本研究中，對於NS3內切反應的作用機制也作了探討，結果顯示即使是保有作為NS3 serine protease輔因子活性的NS4A變異株 [NS4A(I25AV26A)]，也不足以使NS3產生內切片段。這顯示內切片段的產生可能不是NS3具有serine protease的活性即可達成的，這點與Yang等人觀點不相同。此一研究亦顯示NS4A第25個位置之Isoleucine及/或第26個位置之Valine，對於NS3能否被內切具有關鍵性的角色。此外，Yang等人研究指出NS3的內切片段較完整的NS3具有更高的轉型能力，於是本研究利用soft agar assay比較NS4A或NS4A(I25AV26A) 存在時對於NS3轉型能力的影響。結果顯示，相較於其他實驗組，NS4A與NS3存在下，細胞轉型的比例最高。但由於長出的聚落太少，誤差值較大，且其中轉染v-Src的正控制組其細胞轉型的聚落數偏低，因此有關於NS4A對NS3轉型能力的影響仍需作進一步的確認。了解HCV各個蛋白質之間的相互作用，相信對於HCV致病機制上能有更深一層的了解。

關鍵字

C型肝炎病毒 ； 非結構性蛋白質4A ； 轉譯 ； 非結構性蛋白質3 ； 內部截切 ； 轉型

並列摘要

Hepatitis C virus (HCV) is a positive, single-stranded RNA virus. Previous studies have demonstrated that the viral nonstructural protein NS4A interacts with NS3 and is a cofactor of NS3 serine protease essential for the proteolytic processing of the viral polyprotein. NS4A protein was also demonstrated to inhibit cellular and viral protein synthesis. By performing GST pull down assay, our laboratory has previously identified eEF1A that specifically interacted with NS4A. The purpose of this study was to examine whether NS4A inhibits protein translation via interacting with eEF1A. By performing luciferase assay, the inhibition of translation by NS4A protein was confirmed in this study. The eEF1A C-terminal domain, responsible for binding with eEF1B and tRNA was identified to be involved in the interaction. Furthermore, mutations at Val-23 [NS4A(V23A)] and at Ile-25 and Val-26 [NS4A(I25AV26A)] disrupted the interaction between NS4A and eEF1A. The effect of NS4A on translation inhibition was also decreased with V23 and I25AV26A mutations. In contrast, a mutation at Val-24 [NS4A(V24A)] remained the ability of NS4A to interact with eEF1A and the inhibition effect on luciferase activity. These results suggest NS4A may interfere with protein synthesis by forming complex with eEF1A. This may be involved in HCV infection and helps its survival in host cells.
It was reported the NS3 protein is internally cleaved in the presence of NS4A when expressed in HepG2, COS-7, and NIH3T3 cells. The internal cleavage of NS3 protein required not only NS4A as a cofactor but also the activity of NS3 serine protease. However, in this study, we found that NS4A(I25AV26A) that retains the function to act as a cofactor of NS3 was unable to induce the internal cleavage of NS3. It suggests that the serine protease activity of NS3 is not sufficient for its internal cleavage. In addition, the internal cleavage products of NS3 appeared to have higher oncogenic potential than the intact NS3. In this study, soft agar analysis was performed to compare the transformation activity of NS3 in the presence of NS4A or NS4A(I25AV26A). Preliminary data did indicate a higher transformation activity of NS3 in the presence of wild type NS4A. Nevertheless, the numbers of transforming colonies were low and may need to be further confirmed. The possible roles of the interactions among the viral nonstructural proteins, on the oncogenesis of HCV infection remain to be elucidated.

並列關鍵字

HCV ； NS4A ； translation ； NS3 ； internal cleavage ； transformation

參考文獻

Santolini E, Migliaccio G, La Monica N. 1994. Biosynthesis and biochemical properties of the hepatitis C virus core protein. J Virol 68:3631-41.

Choo QL, Kuo G, Weiner AJ, Overby LR, Bradley DW, and Houghton M. 1989. Isolation of a cDNA clone derived from a blood-borne non-A, non-B viral hepatitis genome. Science 244:359-62.

Cimarelli A, and Luban J. 1999. Translation elongation factor 1-alpha interacts specifically with the human immunodeficiency virus type 1 Gag polyprotein. J Virol 73:5388-401.

Condeelis J. 1995. Elongation factor 1 alpha, translation and the cytoskeleton. Trends Biochem Sci 20:169-70.

Darke PL, Jacobs AR, Waxman L, Kuo LC. 1999. Inhibition of hepatitis C virus NS2/3 processing by NS4A peptides. Implications for control of viral processing. J Biol Chem 274:34511-4.